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1.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361731

RESUMO

Strigolactones (SLs) are a class of sesquiterpenoid plant hormones that play a role in the response of plants to various biotic and abiotic stresses. When released into the rhizosphere, they are perceived by both beneficial symbiotic mycorrhizal fungi and parasitic plants. Due to their multiple roles, SLs are potentially interesting agricultural targets. Indeed, the use of SLs as agrochemicals can favor sustainable agriculture via multiple mechanisms, including shaping root architecture, promoting ideal branching, stimulating nutrient assimilation, controlling parasitic weeds, mitigating drought and enhancing mycorrhization. Moreover, over the last few years, a number of studies have shed light onto the effects exerted by SLs on human cells and on their possible applications in medicine. For example, SLs have been demonstrated to play a key role in the control of pathways related to apoptosis and inflammation. The elucidation of the molecular mechanisms behind their action has inspired further investigations into their effects on human cells and their possible uses as anti-cancer and antimicrobial agents.


Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Lactonas/farmacologia , Micorrizas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Plantas/metabolismo , Sesquiterpenos/farmacologia , Adaptação Fisiológica , Agricultura/métodos , Agroquímicos/isolamento & purificação , Agroquímicos/metabolismo , Agroquímicos/farmacologia , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/isolamento & purificação , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Inflamação/prevenção & controle , Lactonas/isolamento & purificação , Lactonas/metabolismo , Micorrizas/química , Neoplasias/tratamento farmacológico , Patentes como Assunto , Reguladores de Crescimento de Plantas/biossíntese , Reguladores de Crescimento de Plantas/isolamento & purificação , Plantas/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/metabolismo , Estresse Fisiológico , Controle de Plantas Daninhas/métodos
2.
Molecules ; 26(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209843

RESUMO

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.


Assuntos
Imidazóis , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1
3.
Chem Sci ; 12(13): 4740-4746, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-34163730

RESUMO

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as "unconventional" therapeutics with precise spatiotemporal control by using light stimuli may open entirely new horizons for innovative therapeutic modalities. Among ROS and RNS, peroxynitrite (ONOO-) plays a dominant role in chemistry and biology in view of its potent oxidizing power and cytotoxic action. We have designed and synthesized a molecular hybrid based on benzophenothiazine as a red light-harvesting antenna joined to an N-nitroso appendage through a flexible spacer. Single photon red light excitation of this molecular construct triggers the release of nitric oxide (˙NO) and simultaneously produces superoxide anions (O2˙-). The diffusion-controlled reaction between these two radical species generates ONOO-, as confirmed by the use of fluorescein-boronate as a highly selective chemical probe. Besides, the red fluorescence of the hybrid allows its tracking in different types of cancer cells where it is well-tolerated in the dark but induces remarkable cell mortality under irradiation with red light in a very low concentration range, with very low light doses (ca. 1 J cm-2). This ONOO- generator activatable by highly biocompatible and tissue penetrating single photon red light can open up intriguing prospects in biomedical research, where precise and spatiotemporally controlled concentrations of ONOO- are required.

4.
ACS Pharmacol Transl Sci ; 4(3): 1079-1095, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34136757

RESUMO

The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ∼30 000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ∼200 virtual screenings of compound libraries on selected protein structures, we redefine the protein's druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

5.
Molecules ; 26(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799482

RESUMO

Flavonoids are plant bioactives that are recognized as hormone-like polyphenols because of their similarity to the endogenous sex steroids 17ß-estradiol and testosterone, and to their estrogen- and androgen-like activity. Most efforts to verify flavonoid binding to nuclear receptors (NRs) and explain their action have been focused on ERα, while less attention has been paid to other nuclear and non-nuclear membrane androgen and estrogen receptors. Here, we investigate six flavonoids (apigenin, genistein, luteolin, naringenin, quercetin, and resveratrol) that are widely present in fruits and vegetables, and often used as replacement therapy in menopause. We performed comparative computational docking simulations to predict their capability of binding nuclear receptors ERα, ERß, ERRß, ERRγ, androgen receptor (AR), and its variant ART877A and membrane receptors for androgens, i.e., ZIP9, GPRC6A, OXER1, TRPM8, and estrogens, i.e., G Protein-Coupled Estrogen Receptor (GPER). In agreement with data reported in literature, our results suggest that these flavonoids show a relevant degree of complementarity with both estrogen and androgen NR binding sites, likely triggering genomic-mediated effects. It is noteworthy that reliable protein-ligand complexes and estimated interaction energies were also obtained for some suggested estrogen and androgen membrane receptors, indicating that flavonoids could also exert non-genomic actions. Further investigations are needed to clarify flavonoid multiple genomic and non-genomic effects. Caution in their administration could be necessary, until the safe assumption of these natural molecules that are largely present in food is assured.


Assuntos
Androgênios/metabolismo , Núcleo Celular/metabolismo , Estrogênios/metabolismo , Flavonoides/metabolismo , Ligação Proteica/fisiologia , Receptores de Superfície Celular/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Humanos , Simulação de Acoplamento Molecular , Receptores de Estrogênio , Testosterona/metabolismo
6.
J Med Chem ; 64(6): 3204-3221, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33710891

RESUMO

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Ácido Fólico/análogos & derivados , Peptídeos/química , Peptídeos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Antineoplásicos/farmacocinética , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Receptor 1 de Folato/metabolismo , Ácido Fólico/farmacocinética , Ácido Fólico/farmacologia , Humanos , Modelos Moleculares , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Peptídeos/farmacocinética , Timidilato Sintase/metabolismo
7.
FEBS J ; 288(9): 3034-3054, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33249721

RESUMO

Human serine racemase (hSR) catalyzes the biosynthesis of D-serine, an obligatory co-agonist of the NMDA receptors. It was previously found that the reversible S-nitrosylation of Cys113 reduces hSR activity. Here, we show by site-directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S-nitrosylation stabilizes an open, less-active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation-dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the ε-amino group of Lys114 acts as a base toward the thiol group of Cys113. In the closed conformation stabilized by glycine-an active-site ligand of hSR-the side chain of Lys114 moves away from that of Cys113, while the carboxyl side-chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S-nitrosylation constitute a three-way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2 O2 -mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.


Assuntos
Regulação Alostérica/genética , Conformação Proteica , Racemases e Epimerases/ultraestrutura , Sítios de Ligação , Domínio Catalítico/genética , Glicina/genética , Humanos , Cinética , Oxirredução , Racemases e Epimerases/química , Racemases e Epimerases/genética
8.
Antibiotics (Basel) ; 9(11)2020 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-33233339

RESUMO

ß-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics.

9.
Sensors (Basel) ; 20(17)2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887407

RESUMO

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Staphylococcus aureus , Peptídeos , Staphylococcus aureus/isolamento & purificação
10.
Sci Rep ; 10(1): 12763, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728062

RESUMO

Bacteria are known to evade ß-lactam antibiotic action by producing ß-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available ß-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Serina/química , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/farmacologia , Cristalografia por Raios X , Bases de Dados de Proteínas , Desenho de Fármacos , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Hidrólise , Ligantes , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Semicarbazidas/química , Compostos de Sulfidrila/química , Sulfonamidas/química , Tetrazóis/química , beta-Lactamases
11.
Nanomedicine ; 28: 102226, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32479916

RESUMO

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood-brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models.


Assuntos
Nanomedicina/métodos , Peptídeos/química , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistema Nervoso Central , Sistemas de Liberação de Medicamentos/métodos , Humanos , Oligopeptídeos/química
12.
Microorganisms ; 8(5)2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-32397638

RESUMO

The human cytomegalovirus (HCMV) is a widespread pathogen and is associated with severe diseases in immunocompromised individuals. Moreover, HCMV infection is the most frequent cause of congenital malformation in developed countries. Although nucleoside analogs have been successfully employed against HCMV, their use is hampered by the occurrence of serious side effects. There is thus an urgent clinical need for less toxic, but highly effective, antiviral drugs. Strigolactones (SLs) are a novel class of plant hormones with a multifaceted activity. While their role in plant-related fields has been extensively explored, their effects on human cells and their potential applications in medicine are far from being fully exploited. In particular, their antiviral activity has never been investigated. In the present study, a panel of SL analogs has been assessed for antiviral activity against HCMV. We demonstrate that TH-EGO and EDOT-EGO significantly inhibit HCMV replication in vitro, impairing late protein expression. Moreover, we show that the SL-dependent induction of apoptosis in HCMV-infected cells is a contributing mechanism to SL antiviral properties. Overall, our results indicate that SLs may be a promising alternative to nucleoside analogs for the treatment of HCMV infections.

13.
Chem Commun (Camb) ; 56(47): 6332-6335, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32435776

RESUMO

We report for the first time a NO photodonor (NOPD) operating with the widely used chemotherapeutic agent doxorubicin (DOX) as the light-harvesting antenna. This permits NO uncaging from an N-nitroso appendage upon selective excitation of DOX with highly biocompatible green light, without precluding its typical red emission. This NOPD effectively binds DNA and photodelivers NO nearby, representing an intriguing candidate for potential multimodal therapeutic applications based on the combination of DOX and NO.


Assuntos
Antineoplásicos/química , Materiais Biocompatíveis/química , Doxorrubicina/química , Luz , Óxido Nítrico/química , Humanos , Estrutura Molecular
14.
Chemistry ; 26(60): 13627-13633, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-32453464

RESUMO

A novel molecular hybrid has been designed and synthesized in which acridine orange (AO) is covalently linked to an N-nitrosoaniline derivative through an alkyl spacer. Photoexcitation of the AO antenna with the highly biocompatible green light results in intense fluorescence emission and triggers NO detachment from the N-nitroso appendage via an intramolecular electron transfer. The presence of the AO moiety encourages the binding with DNA through both external and partially intercalative fashions, depending on the DNA:molecular hybrid molar ratio. Importantly, this dual-mode binding interaction with the biopolymer does not preclude the NO photoreleasing performances of the molecular hybrid, permitting NO to be photogenerated nearby DNA with an efficiency similar to that of the free molecule. These properties make the presented compound an intriguing candidate for fundamental and potential applicative research studies where NO delivery in the DNA proximity precisely regulated by harmless green light is required.


Assuntos
Laranja de Acridina , DNA , Óxido Nítrico , Corantes Fluorescentes , Luz , Nitrosaminas , Processos Fotoquímicos , Espectrometria de Fluorescência
15.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32245010

RESUMO

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery.


Assuntos
Antibacterianos/farmacologia , Interações Hospedeiro-Patógeno , Imunidade , Ferro/metabolismo , Fenômenos Fisiológicos da Nutrição , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Virulência/efeitos dos fármacos
16.
Pharmaceuticals (Basel) ; 13(3)2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32213902

RESUMO

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available ß-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of ß-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine ß-Lactamase (SBLs) KPC-2 and class B1 metallo ß-Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

17.
Cancer Lett ; 470: 115-125, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31693922

RESUMO

Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Oncogênicas Virais/antagonistas & inibidores , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Cristalografia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Neoplasias/patologia , Neoplasias/virologia , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Esferoides Celulares , Relação Estrutura-Atividade
18.
Biochimie ; 170: 106-117, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31887335

RESUMO

Cholesterol biosynthesis is a multistep process in mammals that includes the aerobic removal of three methyl groups from the intermediate lanosterol, one from position 14 and two from position 4. During the demethylations at position 4, a 3-ketosteroid reductase catalyses the conversion of both 4-methylzymosterone and zymosterone to 4-methylzymosterol and zymosterol, respectively, restoring the alcoholic function of lanosterol, which is also maintained in cholesterol. Unlike other eukaryotes, mammals also use the same enzyme as an estrone reductase that can transform estrone (E1) into estradiol (E2). This enzyme, named 17ß-hydroxysteroid dehydrogenase type 7 (HSD17B7), is therefore a multifunctional protein in mammals, and one that belongs to both the HSD17B family, which is involved in steroid-hormone metabolism, and to the family of post-squalene cholesterol biosynthesis enzymes. In the present study, a series of known inhibitors of human HSD17B7's E1-reductase activity have been assayed for potential inhibition against 3-ketosteroid reductase activity. Surprisingly, the assayed compounds lost their inhibition activity when tested in HepG2 cells that were incubated with radiolabelled acetate and against the recombinant overexpressed human enzyme incubated with 4-methylzymosterone (both radiolabelled and not). Preliminary kinetic analyses suggest a mixed or non-competitive inhibition on the E1-reductase activity, which is in agreement with Molecular Dynamics simulations. These results raise questions about the mechanism(s) of action of these possible inhibitors, the enzyme dynamic regulation and the interplay between the two activities.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Estradiol/metabolismo , Estrona/metabolismo , Proteína A6 Ligante de Cálcio S100/antagonistas & inibidores , Proteína A6 Ligante de Cálcio S100/metabolismo , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Colesterol/metabolismo , Inibidores Enzimáticos/química , Estrogênios/metabolismo , Células Hep G2 , Humanos , Conformação Proteica
19.
Life Sci ; 241: 117116, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31790690

RESUMO

AIMS: LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage. MAIN METHODS: The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis was applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation. KEY FINDINGS: Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation. SIGNIFICANCE: The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process toward effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Compostos de Boro/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Compostos de Boro/química , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Levofloxacino/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Resposta SOS em Genética/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/genética
20.
Sci Rep ; 9(1): 18629, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819099

RESUMO

Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and ß subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb.


Assuntos
Proteínas de Transporte de Cátions/genética , Hemoglobinas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/genética , Resistência a Múltiplos Medicamentos/genética , Heme/genética , Humanos , Ferro/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Infecções Estafilocócicas/microbiologia
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